Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression
© Joshi et al; licensee BioMed Central Ltd. 2011
Received: 12 January 2011
Accepted: 28 June 2011
Published: 28 June 2011
The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown.
We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells.
Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.
Drugs that disrupt mitotic progression are commonly referred to as 'anti-mitotics' and are extensively used for the treatment of cancer. Classical 'anti-mitotic' chemotherapeutics used in the clinic target microtubules and include the taxanes and vinca alkaloids . Despite success in the clinic, drug resistance and toxicity have limited their effectiveness, due to the broad role of tubulin in the cytoskeleton of normal and non-dividing cells . A new class of anti-mitotics have been developed that specifically target mitotic proteins such as Aurora kinase, polo-like kinase, kinesin spindle protein [1, 2]. Such inhibitors are being characterised as potential chemotherapeutic agents since several induce mitotic failure leading to apoptotic cell death in cancer cells and xenograft mouse cancer models [2, 3]. These mitotic proteins are either expressed only in dividing cells or apparently function exclusively during mitosis. In contrast to classical anti-mitotics, non-dividing differentiated cells should not be affected by such targeted inhibition, and thus they are predicted to be more efficacious. Many of these targeted inhibitors are currently in cancer clinical trials. Despite the differences in the protein being targeted, both classical and targeted anti-mitotics developed to date aim to disrupt the mitotic spindle or an early stage in mitosis.
We have recently reported a new class of targeted anti-mitotics that do not perturb the mitotic spindle but exclusively block cytokinesis . The targeted protein for inhibition is the endocytic protein, dynamin II (dynII). DynII is best known for its role in membrane trafficking processes, specifically in clathrin-mediated endocytosis [5–7]. However, dynII also plays an essential role in the completion of the final stage of mitosis, cytokinesis [4–6, 8–12]. We and others have developed several classes of dynamin inhibitors including dynasore , dimeric tyrphostins (Bis-Ts), long chain amines and ammonium salts (MiTMABs (myristyl trimethyl ammonium bromides)), dynoles [14–16], iminodyns  and pthaladyns . Characterisation of the two most potent MiTMABs, MiTMAB and OcTMAB (collectively referred to as MiTMABs), revealed that they block the abscission phase of cytokinesis causing polyploidization, which is analogous to the dynII siRNA phenotype [4, 8]. The MiTMAB dynamin inhibitors share many favourable characteristics with inhibitors of Aurora kinases, Plk and KSP: (i) they do not affect any other phase of the cell division cycle and (ii) possess anti-proliferative and cytotoxic properties that are selective for cancer cells . Thus, targeting cytokinesis with dynamin inhibitors may be a promising new approach for the treatment of cancer.
Apoptotic cell death is central to targeted anti-mitotic compounds being highly efficacious as chemotherapeutic agents and is thought to depend on their ability to cause mitotic failure and subsequent accumulation of polyploid cells [3, 19–21]. The mechanism of apoptosis following mitosis failure is poorly understood. It is thought to be classical apoptosis, involving caspase activation and poly(ADP-ribose) polymerase 1 (PARP1) cleavage . However, cell death induced by caspase-independent mechanisms has been reported [23, 24]. Apoptotic cell death does not always result following mitotic failure induced by an anti-mitotic. Various cellular responses, depending on the cell line and inhibitor analysed have been reported and include apoptosis, senescence and reversible mitotic arrest . An in-depth understanding of the mechanisms driving a particular cellular fate in response to targeted anti-mitotics is crucial for rational development and their potential application as chemotherapeutic agents.
In this study, we aimed to determine the fate of cells and the signalling mechanisms involved following treatment with MiTMABs, which exclusively block abscission during cytokinesis. We report that MiTMABs induce cell death following cytokinesis failure in several cancer cells and this was mediated by the intrinsic apoptotic pathway. The cellular response of cancer cells to MiTMABs appeared to correlate with expression of Bcl-2. Our results indicate that the anti-proliferative and cytotoxic properties of the MiTMAB dynamin inhibitors are due to their ability to induce apoptosis following cytokinesis failure. This provides the first evidence that targeting cytokinesis is a valid approach for the development of anti-cancer agents, and that dynII inhibitors are the first class of compounds in this new targeted anti-mitotic group.
HeLa, HeLa-Bcl-2  and H460 cell lines were maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) and 5% (P/S). HT29, SW480 and MCF-7 cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and 5% P/S. All cells were grown at 37 °C in a humidified 5% CO2 atmosphere.
The active dynamin inhibitors, MiTMAB (also known as tetradecyl trimethyl ammonium bromide, CAS number 119-97-7), OcTMAB (CAS number 1120-02-1; Sigma-Aldrich, Co., St. Louis, MO), and the inactive analogue, 2-(DiMA)EM (2-(dimethylamino) ethyl myristate; Lancaster Synthesis, England), were prepared as 30 mM stock solutions in DMSO and stored at -20°C. Cytochalasin B (cytB) was prepared as 5 mg/ml stock solutions in DMSO and stored at -20°C. The CDK1 small molecule inhibitor RO-3306 was synthesised in-house as reported previously . Stock solution (9 mM) of RO-3306 was prepared in DMSO and stored at -20°C. The pan-caspase inhibitor Z-VAD-FMK (ZVAD) and the caspase-8 selective inhibitor Z-IETD-FMK (IETD) were purchased from BD Biosciences and used at a final concentration of 50 µM.
Cell synchronization and treatment with MiTMABs
Cells were synchronized at the G2/M boundary by treatment with RO-3306 (9 µM) for 18 hours [4, 8] and at the G1/S boundary by the double thymidine block assay  as previously described. Immediately following RO-3306 or thymidine removal, cells synchronously entered the cell cycle and were treated with MiTMABs. As a negative control, cells were released into drug-free medium, or medium containing 0.1% DMSO or the inactive analogue 2-(DiMA)EM. As a positive control for apoptosis, cells were irradiated with ultraviolet (UV-C) light at 100 J/m2.
Cell cycle analysis by flow cytometry
Cells (5 × 105 cells per dish) were grown in 10 cm dishes. Following inhibitor treatment, cells (floating and adherent) were collected and single-cell suspensions were fixed in 80% ice-cold ethanol at -20 °C for at least 16 hours. Cells were stained with propidium iodide and cell cycle was analysed . Cell cycle profiles were acquired with a FACS Canto Flow Cytometer (Becton Dickinson) using FACS Diva software (v.5.0.1) at 488 nm. Cell cycle profiles were analysed using FlowJo software (v.7.1).
Where indicated, the drugs were removed by washing three times with drug-free medium after a 6 h treatment. Cells were then incubated for an additional 42 h in drug-free medium prior to fixation and flow cytometry analysis.
Cells were seeded in 6-well plates (1 × 105 cells per well) and synchronized at the G2/M boundary as described above. Immediately following release into the cell cycle, cells were treated with the indicated molecule and viewed with an Olympus IX80 inverted microscope. A time-lapse series was acquired using a fully motorised stage, 10x objective, and Metamorph software using the time-lapse modules. Temperature was controlled at 37 °C using the Incubator XL, providing a humidified atmosphere with 5% CO2. Images were captured every 10 minutes for 20 hours. Where indicated, a time-lapse series was acquired in asynchronously growing cells immediately following the addition of the indicated drug.
Cells were fixed in ice-cold 100% methanol and immunostaining was carried using the anti-α-tubulin (Clone DM1A; Sigma) antibody [4, 27]. Cells were viewed and scored for multinucleation with a fluorescence microscope (Olympus BX51). Fluorescence images were captured and processed using an Olympus IX80 inverted microscope using 40x or 100x oil immersion lenses and Metamorph software. Images were deconvolved using AutoDeblur v.9.3 (AutoQuant Imaging, Watervliet, NY).
Cell lysates were prepared as described previously . In brief, cells were collected by centrifugation, washed with PBS, then resuspended in ice-cold lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 and EDTA-free Complete protease inhibitor cocktail (Roche)) for 30 mins. The supernatant (cell lysate) was collected following centrifugation at 13,000 rpm for 30 min at 4ºC. Cell lysates (50 μg) were fractionated by SDS-PAGE for immunoblot analysis using the following primary antibodies: Bcl-2, Bcl-XL, Mcl-1, cleaved caspase-8, -9, -3, PARP (Cell Signaling Tech) and β-actin (Sigma-Aldrich). Primary antibody was detected by incubation with horseradish peroxidise-conjugated anti-rabbit or anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories). Blotted proteins were visualized using the ECL chemiluminescence detection system (Pierce).
HeLa cells undergo apoptosis following cytokinesis failure
We next sought to determine when after cytokinesis failure the cells were committed to apoptosis by using flow cytometry. By 6 h after release from the G2/M boundary, the majority of cells have entered mitosis and completed this process albeit either successfully (two mononucleated daughter cells) or unsuccessfully (binucleated cell). At this time point, no morphological signs of apoptosis are evident. As expected, after a 48 h treatment period, OcTMAB induced apoptosis in G2/M synchronized cells, as evident by an increase in the percentage of cells with <2N DNA content (Figure 1D). Apoptosis was still evident in cells after 48 h when OcTMAB was removed by wash-out after only a short 6 h treatment (Figure 1D), indicating that the cells were already committed to cell death very soon after cytokinesis failure and binucleate formation. This again suggests that the induction of apoptosis is associated with cytokinesis failure and not due to generalised toxicity of the MiTMABs.
HeLa cells undergo caspase-mediated apoptosis exclusively following cytokinesis failure
HeLa cells stably expressing Bcl-2 are resistant to MiTMABs-induced cell death
MiTMABs-induced cell death occurs via the intrinsic apoptotic pathway
The apoptotic response of cancer cells to MiTMABs appears to correlate with expression of Bcl-2 and Mcl-1 anti-apoptotic proteins
We next sought to gain insight into why specific cancer cells are sensitive (HeLa, SW480 and HT29) and others are resistant (H460 and MCF-7) to apoptosis induced by MiTMABs. We showed that HeLa cells stably expressing the anti-apoptotic protein, Bcl-2, are resistant to apoptosis induced by MiTMABs. Moreover, Bcl-2 family members are frequently over-expressed in cancers and confer resistance to anti-mitotic chemotherapy in various tumour types [37, 38]. Therefore, we analysed the expression levels of three anti-apoptotic Bcl-2 family members, Bcl-2, Bcl-XL and Mcl-1, in all five cancer cell lines. Immunoblotting revealed that the three lines which are sensitive to MiTMABs, HeLa, HT29 and SW480, have relatively low levels of Bcl-2 and Mcl-1 (Figure 7C), which correlated well with the ability of MiTMABs to induce apoptosis in these cells. Although the MiTMABs-resistant MCF-7 cells also expressed low levels of these proteins (Figure 7C), their resistance can likely be explained by their underlying deficiency in caspase-3 . In contrast, high levels of Bcl-2 and Mcl-1 proteins were detected in H460 cells (Figure 7C). Again, this correlated well with resistance of this cell line to MiTMABs-induced apoptosis. Except for HeLa cells, which expressed almost undetectable levels of Bcl-XL, the other four cell lines expressed moderate levels (Figure 7C). Thus, unlike Bcl-2 and Mcl-1, Bcl-XL protein levels did not correlate well with sensitivity to MiTMABs. The results suggest that the ability of MiTMABs to induce apoptosis appears to be dependent on the relative expression levels of the anti-apoptotic proteins Bcl-2 and Mcl-1.
Dynamin inhibitors are a new class of targeted anti-mitotic compounds. In contrast to the classical (e.g. taxol) and known targeted (e.g. Aurora kinase and Plk inhibitors) anti-mitotic compounds which aim to disrupt the mitotic spindle, the MiTMAB dynamin inhibitors exclusively block cytokinesis without disrupting progression through any other stage of mitosis. Analogous to other anti-mitotic compounds, dynamin inhibitors also have putative anti-tumour activity . In this study, we show that two dynamin inhibitors called the MiTMABs induce cytokinesis failure and induce apoptosis in cancer cells and this appears to correlate with low expression of the anti-apoptotic proteins Bcl-2 and Mcl-1. Apoptosis occurred strictly following formation of a polyploid cell and was mediated via the intrinsic pathway. Over-expression of the anti-apoptotic protein, Bcl-2, blocked MiTMAB-induced apoptosis but not polyploidization. The induction of apoptosis exclusively following mitotic damage is analogous to the effect of targeted anti-mitotics, such as aurora kinase and Plk inhibitors . We also demonstrate that apoptosis is induced in cells that have failed cytokinesis due to treatment with the cytokinesis blocker, cytochalsin B. Therefore, this is the first study to demonstrate that cytokinesis blockers can specifically induce apoptotic cell death and thus represent a new class of anti-mitotics with potential anti-cancer activity. Our results indicate that dynamin II is the primary target in this new anti-mitotic action.
Cells exposed to MiTMAB undergo cell death via activation of the intrinsic apoptotic pathway. This was evident by the presence of cleaved caspase-3, -9, and PARP, an increase in DNA fragmentation (<2N DNA content), and membrane blebbing. We further demonstrate that this intrinsic apoptotic pathway involves a feedback caspase-8 amplification loop to drive the execution of apoptosis. MiTMAB-induced cell death exclusively occurred following cytokinesis failure and subsequent polyploidization. This was demonstrated by several findings. Independent single cell analysis using time-lapse microscopy revealed that those MiTMAB-treated cells that failed cytokinesis subsequently underwent apoptotic cell death. We observed an increase in polyploidization in MiTMAB-treated cells when apoptosis was blocked by ZVAD or Bcl-2 overexpression. Caspase-8, -9, -3 and PARP cleavage products were not observed in cells treated with MiTMABs that were not able to undergo a mitotic division (8 h treatment from G1/S synchronization). Similar reports of cell death specifically following polyploidization in the presence of targeted inhibitors, such as aurora kinase, Plk and KSP inhibitors, have been reported [1, 2, 40]. This indicates that inhibition of a specific target is not the trigger for apoptosis but rather that it is the phenotype or subsequent molecular alteration generated as a result of its disruption.
The ability of anti-mitotic compounds to induce apoptosis exclusively in dividing cells is the primary rationale that they may be efficacious chemotherapeutic compounds [3, 19, 20, 41]. However, an increased level of polyploidization does not appear to translate into increased level of secondary apoptosis . Rather the resulting induction of apoptosis appears to be cell type specific. In line with this idea, the cellular response following exposure to a particular anti-mitotic varies and includes not only apoptosis, but also mitotic catastrophe, senescence and reversible mitotic arrest . One determinant thought to predict the cellular response to a particular anti-mitotic is the time spent blocked in mitosis . In the presence of the microtubule-stabilising drugs, ZM447439 (Aurora A/B inhibitor) and taxol, cells blocked in mitosis for >15 h undergo apoptosis shortly after mitotic exit, whereas those cells blocked in mitosis for <15 h showed variable fates with some cells living for days after mitotic exit . This analysis was carried out in HeLa cells, as done in the present study. In contrast to these findings, the MiTMABs, which block cytokinesis, did not trap cells at this mitotic stage for a long period of time, but only slightly delayed mitotic exit by approximately 30 mins . Nevertheless, time-lapse analysis indicated that every MiTMAB treated HeLa cell failing cytokinesis proceeded to apoptotic cell death approximately 7-10 hours after exiting mitosis. Conversely, we have previously shown that H460 cells spend a prolonged period of time trapped in cytokinesis in the presence of MiTMABs (up to 24 h)  and these cells remained viable during the following 24 h time period of analysis. Thus, in the case of the MiTMAB-based dynamin inhibitors, the induction of apoptosis appears to correlate with a short (rather than long) period of time that cells spend trapped in cytokinesis. The significance of this correlation needs to be investigated in more detail. Rather, the difference in apoptotic response between these two cell lines likely represents the underlying difference in their molecular components, such as p53 status and Bcl-2 protein levels.
Several reports suggest that p53 status is critical for determining the cellular response following polyploidization [21, 44, 45]. It is possible that MiTMAB-induced cell death is influenced by p53 status since its expression or mutation status also correlated with sensitivity (HeLa: p53wt but almost undetectable levels due to HPV, HT29: p53mut and SW480: p53mut) and resistance (MCF-7 and H460 contain p53wt) to apoptosis. Given that this gene is frequently lost or mutated in cancers , the ability of dynamin inhibitors to induce apoptosis following polyploidization in cells lacking functional p53 could be a favourable characteristic as a potential chemotherapeutic agent. It could be particularly relevant to those drug resistant cancers that often develop following p53 mutation. However, the contribution of p53 in determining the cellular response following polyploidization is under debate and is complicated by its multiple roles. For example, in response to aurora kinase inhibitors, p53wt is required for G1 arrest of tetraploid cells  and for inducing apoptosis following tetraploid formation . Therefore, p53 status alone is not the sole predictor of the cellular response following polyploidization.
The expression of Bcl-2 and Mcl-1, but not Bcl-XL, appears to correlate with the ability of cells to undergo apoptosis following exposure to MiTMABs. There are six anti-apoptotic Bcl-2 family members identified and several of these appear to contribute to drug resistance in cancer cells [37, 38], suggesting that inhibition of multiple Bcl-2 family members will be necessary to achieve an optimal therapeutic effect. The development of antagonists toward Bcl-2  and Mcl-1  provide an attractive hypothesis that MiTMABs may synergise with these antagonists to sensitise resistant cell lines to undergo apoptosis. In line with this idea, the Bcl-2 antagonists, ABT-737 or ABT-263, have been shown to synergise with Plk and aurora kinase inhibitors  as well as conventional chemotherapeutic drugs, such as vincristine, in vitro and in vivo.
Overall, our findings demonstrate that the MiTMAB family of dynamin inhibitors induce apoptosis in a concentration-dependent manner following polyploidization. More specifically, these are the first reported targeted anti-mitotic compounds which induce polyploidy by exclusively blocking cytokinesis. Thus, dynamin inhibitors are a new class of anti-mitotic compounds with potential anti-cancer action. MiTMAB-induced apoptosis is not only dependent on cytokinesis failure and polyploidization but also on specific molecular components of the apoptotic machinery, such as Bcl-2. Thus, inhibitors of these anti-apoptotic proteins, such as the Bcl-2 inhibitor ABT-737, may act synergistically with the MiTMAB dynamin inhibitors, broadening their therapeutic potential for the treatment of cancer.
We wish to thank Christine Smyth and Swetha Perera for technical assistance. This work was supported by grants from the National Health and Medical Research Council (NH&MRC) of Australia (P.J.R. and M.C.), New South Wales Cancer Council (M.C.) and the NH&MRC Career Development Award (M.C.). A.W.B. is supported by Leader's Fellowship from the Cancer Institute, New South Wales.
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